逆转录聚合酶链反应检测石蜡包埋组织中细胞周期蛋白D1 mRNA对套细胞淋巴瘤的诊断价值

目的 探讨用逆转录聚合酶链反应（RT-PCR）法和竞争性RT-PCR法检测套细胞淋巴瘤（MCL）石蜡包埋组织中细胞周期蛋白D1（cyclin D1）蛋白和mRNA在常规病理工作中的可行性及其诊断和鉴别诊断价值。方法 收集淋巴结内MCL38例、对照组包括结内小B细胞淋巴瘤58例（B小淋巴细胞性淋巴瘤14例,淋巴浆细胞性淋巴瘤3例,滤泡性淋巴34例,淋巴结边缘区B细胞淋巴瘤7例）和淋巴结反应性增生病例20例,用免疫组织化学EnVision法和RT-PCR法、竞争性RT-PCR法检测cyclin D1蛋白及其mRNA的表达,以看家基因PGK作为内对照检测RNA。结果 （1）38例结内MCL中,cyclin D1蛋白阳性率为71．1％（27／38）,对照组均为阴性。（2）116例标本中,可检出内对照PGK基因mRNA表达103例（88．8％）。38例MCL中PGK阳性36例（94．7％）。（3）38例结内MCL中,34例可检出cyclin D1 mRNA表达,去除PGK和cyclin D1 mRNA均阴性的2例,MCL中cyclin D1 mRNA表达的阳性率为94．4％（34／36）。对照组中B小淋巴细胞性淋巴瘤1例检出cyclin D1 mRNA表达,其余病例均未检出cyclin D1 mRNA表达。PCR结果全部经测序证实。（4）用竞争性RT-PCR,38例结内MCL中27例可检出cyclin D1 mRNA高表达,去除2例PGK也为阴性的病例,MCL中cyclin D1 mRNA高表达率为75．0％（27／36）。对照组小B细胞恶性淋巴瘤及淋巴结反应性增生无1例有cyclin D1 mRNA高表达。结论 RT-PCR方法和竞争性RT-PCR方法可在石蜡包埋组织中检测cyclin D1 mRNA的表达,均可用于MCL的诊断。     

Multiparameter immunohistochemical analysis of the cell cycle proteins cyclin D1, Ki-67, p21WAF1, p27KIP1, and p53 in mantle cell lymphoma

BACKGROUND: Mantle cell lymphoma (MCL) is characterized by overexpression of cyclin D1, a G1 cyclin that participates in the control of cell cycle progression at the G1 to S phase transition. In addition to cyclin D1, other cell cycle regulatory molecules may be involved in the proliferation and progression of MCL. Mutation of p53, deletion of p16(INK4a), and loss of p21(WAF1) expression have been reported in some cases of blastoid MCL. OBJECTIVE: We sought to examine levels of expression of these proteins in typical and blastoid MCL and to determine whether differences were present between these subtypes of lymphomas. DESIGN: A retrospective series of typical and blastoid MCLs was evaluated for expression of the cell cycle-related proteins cyclin D1, p21(WAF1), p27(KIP1), Ki-67, and p53, as well as mitotic index. Paraffin-embedded archival tissues from 24 MCL specimens (17 typical, 7 blastoid) were immunostained with antibodies to p21(WAF1), p27(KIP1), p53, Ki-67, and cyclin D1. The percentage of positive cells for each specimen was estimated by counting 1500 cells under oil immersion microscopy. Levels of antigen expression were compared for the typical and blastoid MCLs. The mitotic index was estimated using twenty 100x oil immersion fields (OIFs) for each specimen. RESULTS: Cyclin D1 expression was seen in 22/24 specimens (92%). Blastoid MCLs were characterized by a significantly higher mean mitotic index (>20 mitoses/20 OIFs) and Ki-67 index (>45%) when compared with typical MCLs (P <.001 and P <.008, respectively; Fisher's exact test). High expression of p27(KIP1) (>25% staining) was seen more frequently in typical MCLs than in the blastoid variants (P =.03; Fisher's exact test). No significant differences were found between typical and blastoid MCLs for the expression of p21(WAF1) or p53. CONCLUSIONS: A significantly higher mitotic index and Ki-67 index were found in blastoid MCLs as compared with typical MCLs. Low p27(KIP1) expression was associated with the blastoid MCL variant. These findings confirm the high proliferative nature of blastoid MCL and suggest a role for p27(KIP1) in the negative regulation of the cell cycle in MCL.     

Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb.     

bcl-1 REARRANGEMENT AND CYCLIN D1 PROTEIN EXPRESSION IN MANTLE CELL LYMPHOMA

Centrocytic lymphoma, or mantle cell lymphoma (MCL), is characterized by a chromosomal translocation t(11;14) (q13;q32) involving the bcl-1 locus on chromosome 11. Cyclin D1 is a cell-cycle regulatory protein essential for G1–S transition and has been identified as a potential transforming gene affected by the translocation. In this study, 32 cases of MCL were analysed for the bcl-1 rearrangement and cyclin D1 protein expression. In 17 cases, a rearrangement at the major translocation cluster of bcl-1 could be detected. Twenty-four cases exhibited nuclear cyclin D1 expression that was not detectable in other B-cell lymphomas ( n =40) or in normal B-cells. In nine MCL samples, cyclin D1 was expressed without a detectable bcl-1 rearrangement. The detection of a t(11;14) by means of classical cytogenetics in one of these cases, however, may suggest that this discrepancy could be due to chromosomal breakages outside the typical translocation cluster region. In two cases, a bcl-1 rearrangement was not accompanied by cyclin D1 expression. This study provides further evidence that cyclin D1 is involved in the pathogenesis of MCL and can be exploited as a diagnostic marker in the differential diagnosis of B-cell lymphomas and in the identification of MCL.     

D cyclins in CD5+ B-cell lymphoproliferative disorders: cyclin D1 and cyclin D2 identify diagnostic groups and cyclin D1 correlates with ZAP-70 expression in chronic lymphocytic leukemia

We analyzed protein expression of cyclin D1, cyclin D2, and cyclin D3 using high-resolution enzymatic amplification staining and flow cytometry in the neoplastic cells from 80 patients with CD5+ B-cell lymphoproliferative disorders. The D cyclins were expressed differentially in chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), and mantle cell lymphoma (MCL) with strong staining of cyclin D1 and D2 in MCL, strong staining of cyclin D1 but weak staining of cyclin D2 in 4 of 5 PLLs, and low-level staining for both cyclins in most CLLs. No correlation between cyclin D1 and D2 and growth rates or CD38 expression was observed. However, cyclin D1 levels were significantly higher in ZAP-70+ CLL cases, although no association between ZAP-70 and cyclin D2 was detected. The results indicate that flow cytometric analysis of D cyclins may help in classification of CD5+ B-cell lymphoproliferative disorders.     

Cyclin D3 expression in non-Hodgkin lymphoma. Correlation with other cell cycle regulators and clinical features

Cyclin D3 is the most widely expressed D-type cyclin and can be rate limiting for G1/S transition. To study the expression of cyclin D3 in non-Hodgkin lymphoma, samples from 198 previously untreated patients with lymphoma from a prospectively collected, population-based lymphoma registry were analyzed immunohistochemically for cyclin D3 expression. In 43 lymphomas (21.7%), cyclin D3 was overexpressed. T-cell lymphomas more frequently overexpressed cyclin D3 than B-cell lymphomas. Furthermore, cyclin D3-overexpressing indolent lymphomas were associated with higher proliferation rate, higher p21Waf1 expression, lower p27Kip1 expression, and altered p53. Cyclin D3 overexpression identified a subgroup of patients with indolent B-cell lymphoma with adverse clinical features: patients were older, more frequently had "B" symptoms and extranodal involvement, and were more frequently in the high-intermediate or high-risk International Prognostic Index groups. At univariate analysis of indolent lymphomas, cyclin D3 overexpression was associated significantly with poor overall survival and poor relapse-free survival. The statistical significance was retained on multivariate analysis of overall survival and relapse-free survival. Our results suggest that cyclin D3 is expressed differentially among lymphoma subtypes and that overexpression might identify a subpopulation of patients with indolent lymphoma with adverse clinical features and poor outcome.     

Mantle Cell Lymphomas Lack Expression of p27kip1, a Cyclin-Dependent Kinase Inhibitor

p27^Kip1 is a cyclin-dependent kinase inhibitor that regulates the decision to enter S phase or withdraw from the cell cycle. In resting cells, the level of p27^Kip1 provides an inhibitory threshold above which G1 cyclin D/E/cyclin-dependent kinases accumulate before activation; however, in cycling cells, p27^Kip1 protein is sequestered by high levels of active cyclin D/cyclin-dependent kinase 4 complexes. As a group, the cyclin-dependent kinase inhibitors have been proposed to act as tumor suppressor genes, and several members have been implicated in the pathogenesis of a variety of human cancers. We examined p27^Kip1 expression in 116 non-Hodgkin’s lymphomas including 50 cases of MCL (40 typical and 10 blastic variants), 21 follicular lymphomas, 20 diffuse large B-cell lymphomas, 16 chronic lymphocytic leukemias, 8 marginal zone B-cell lymphomas, and 1 splenic marginal zone lymphoma, and correlated its expression with that of the proliferation marker Ki67 (MiB1) and with p53. p27^Kip1gene structure was analyzed by Southern blot in the group of MCLs. In all cases of non-Hodgkin’s lymphoma other than MCL, p27^Kip1 expression was inversely related to the proliferation index as measured by Ki67. In contrast, in typical MCL, p27^Kip1 expression was negative in 35 of 40 (88%) cases, irrespective of the proliferative rate (median 15%; range 2 to 90%). Paradoxically, in the blastic variant of MCL, 8 of 10 (80%) cases showed expression of p27^Kip1, despite a high proliferation rate (median 60%; range 32 to 100%). However, the staining in most of the cases was less intense than in the reactive T lymphocytes. Deletions of p27^Kip1gene were not found in any of the 25 cases examined. p53 expression was found in 15 of 50 cases of MCL: 7 of 10 (70%) in the blastic variant and 8 of 40 (20%) in the typical MCL (70% vs. 20%, P < 0.0045). These results demonstrate that MCLs, in contrast to other non-Hodgkin’s lymphomas and normal lymphoid tissue, fail to correlate p27^Kip1 expression with the proliferation rate. This peculiar uncoupling of p27^Kip1 protein expression from the proliferation rate may be related to the high levels of cyclin D1 expressed in MCL and is likely to have profound effects on cell cycle regulation and contribute to the pathogenesis of MCL.     

Cyclin alterations in giant cell tumor of bone.

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Because alterations of several cyclins, especially cyclin D1, have been implicated in the development of many human neoplasms, we examined 32 cases of giant cell tumor of long bones for cyclin D1 gene amplification and protein overexpression using differential polymerase chain reaction and immunohistochemistry, respectively. In addition, the expression of cyclin D3, cyclin B1, and the proliferation-associated antigen Ki-67 (MIB-1) was assessed immunohistochemically. Low-level cyclin D1 gene amplification was detected in 61% of giant cell tumor cases. All tumors showed cyclin D1, cyclin D3, cyclin B1, and Ki-67 (MIB-1) staining; however, the distribution was very characteristic. Cyclin D1 protein expression was seen predominantly in the nuclei of the giant cells, with occasional mononuclear cells staining. There was no correlation between cyclin D1 gene amplification and protein overexpression. Cyclin D3 staining showed a similar distribution, with 88% of cases showing protein overexpression. Cyclin D1 and/or D3 staining in the giant cells was never associated with staining for either cyclin B1 or Ki-67 (MIB-1), as the expression of the latter two proteins was restricted to the mononuclear cells. Cyclin B1 overexpression was seen in 44% of cases. Ki-67 (MIB-1) staining was present in all cases, and between 10 to 50% of the mononuclear cells were positive. These results suggest that alterations in cyclin D1 and/or D3 might play a role in the pathogenesis of giant cell tumor of bone.     

Reduced or absent cyclin H expression is an independent prognostic marker for poor outcome in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults, accounting for nearly 40% of all non-Hodgkin's lymphomas. As cell proliferation is essential for tumor growth, analysis of the cell cycle might give additional information on tumor progression. Although markers distinctive for cell-cycle regulation in DLBCL have been addressed, less attention has been paid to cyclin H in DLBCL with respect to its prognostic and potential therapeutic implications. Cyclin H occurs as a component of the cyclin H/Cdk 7/Mat 1 complex. Cyclin H is also a substrate of protein kinase 2, a ubiquitously expressed serine/threonine protein kinase required for cell viability and cell-cycle progression. We evaluated the expression of cyclin H by immunohistochemistry in 301 DLBCLs in a tissue microarray format. Validation was done by performing quantitative real-time polymerase chain reaction and Western blotting experiments for cyclin H. We studied the relationship between cyclin H expression in comparison to other cyclins (A, B1, D1, D3, and E) and the proliferation marker Ki-67. Reduced or absent cyclin H expression was seen in 14.5% of the DLBCL cases. Interestingly, reduced or absent cyclin H expression was correlated with lower expression of proliferation marker Ki-67 (P < .0001), cyclin B1 (P = .0001), cyclin D3 (P = .0007), and cyclin E (P < .0001). Reduced or absent cyclin H expression was significantly associated with poor overall survival, in both the univariate (P = .0286) and multivariate analysis with International Prognostic Index (P = .0180). Our study demonstrates the independent prognostic value of cyclin H expression in DLBCL and proposes its use as a prognostic marker.     

Immunohistochemical detection of cyclin D1 using optimized conditions is highly specific for mantle cell lymphoma and hairy cell leukemia.

Mantle cell lymphoma (MCL) is more aggressive when compared with other lymphomas composed of small, mature B lymphocytes. Cyclin D1 is overexpressed in MCL as a result of the translocation t(11;14)(q13;q32). Cyclin D1 immunohistochemistry in fixed, paraffin-embedded tissue contributes to the precise and reproducible diagnosis of MCL without the requirement of fresh tissue. However, its use in bone marrow biopsies is not well established. In addition, increased levels of cyclin D1 mRNA have been found in hairy cell leukemia but have not consistently been detected by immunohistochemistry. We used a polyclonal antibody and heat-induced antigen retrieval conditions to evaluate 73 fixed, paraffin-embedded bone marrow, spleen, and lymph node specimens with small B-cell infiltrates, obtained from 55 patients. Cyclin D1 was overexpressed in 13/13 specimens of MCL (usually strong, diffuse reactivity in most tumor cells) and in 14/14 specimens of hairy cell leukemia (usually weak, in a subpopulation of tumor cells). No reactivity was detected in five cases of B-chronic lymphocytic leukemia; five cases of splenic marginal zone lymphoma; six cases of nodal marginal zone cell lymphoma; two cases of gastric marginal zone cell lymphoma; or ten benign lymphoid infiltrates in bone marrow, spleen, or lymph nodes. In summary, although the total number of studied cases is small and a larger series of cases may be required to confirm our data, we present optimized immunohistochemical conditions for cyclin D1 in fixed, paraffin-embedded tissue that can be useful in distinguishing MCL and hairy cell leukemia from other small B-cell neoplasms and reactive lymphoid infiltrates.     

Prominent intrasinusoidal infiltration of the bone marrow by mantle cell lymphoma

Intrasinusoidal infiltration of bone marrow (BM) may accompany several malignant lymphoproliferative disorders. In small B-cell lymphomas, this pattern is considered specific for splenic marginal zone lymphoma (SMZL) when exclusive or prominent, although it may occur in other subtypes of non-Hodgkin's lymphomas (NHLs) as a minor feature. Here we report 2 cases of mantle cell lymphoma (MCL) with a prominent intrasinusoidal BM infiltration pattern. Both patients presented with massive splenomegaly and peripheral blood involvement characterized by markedly atypical lymphocytes, but no lymphadenopathy. The cytological features and the phenotype of the lymphoma cells were diagnostic of MCL. The malignant B cells showed coexpression of B-cell markers (CD20+ and CD79a+), CD5 antigen, and cyclin D1 by immunohistochemistry. We discuss the specificity of an intrasinusoidal growth pattern in the bone marrow, emphasizing the importance of using a broader immunohistochemical panel in the differential diagnosis of intrasinusoidal BM infiltration by NHL.     

Combined translocation with ZNF198-FGFR1 gene fusion and deletion of potential tumor suppressors in a myeloproliferative disorder

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation, which leads to overexpression of the cyclin D1 (CCND1) gene. This translocation is observed in almost all cases of MCL. In this alteration, the involvement of immunoglobulin heavy chain (IGH) locus plays a key role in the activation of the CCND1 oncogene. Translocations affecting IGH loci are mostly prevalent in B-cell lymphomas, but variant translocations involving immunoglobulin kappa (IGK) or lambda (IGL) light chain loci have been observed in a minority of B-lymphoid malignancies. Variant translocations have been reported in only a few cases of MCL, however. This report presents a case of MCL with a variant t(2;11)(p11.2;q13), rearrangement of the CCND1 gene, and overexpression of cyclin D1. To characterize this rearrangement, specific noncommercial probes were used. This set of probes comprises IGK and REL flanking probes and 12 bacterial artificial chromosome (BAC) probes covering the region to be investigated. The results indicated that this alteration has not affected the IGK locus, and the breakpoint was within a 260-kb region located approximately 1 Mb telomerically to the IGK gene. It is probable that the KV3J gene localized in this region could deregulate the expression of cyclin D1.     

套细胞淋巴瘤石蜡包埋组织中细胞周期蛋白D1和t(11;14)易位检测的临床病理意义

目的 探讨套细胞淋巴瘤石蜡包埋组织中细胞周期蛋白(cyclin)D1和t(11;14)易位检测的可行性及其诊断和鉴别诊断价值。方法 收集套细胞淋巴瘤36例,对照组小B细胞恶性淋巴瘤71例,均为石蜡包埋组织,运用免疫组织化学方法观察cyclin D1的表达;用半巢式聚合酶链反应(PCR)法检测t(11;14)易位,以看家基因β-肌动蛋白(actin)作为内对照检测DNA质量。结果 (1)36例套细胞淋巴瘤中26例(72．2％)表达cyclin D1,对照组无1例表达。(2)107例标本中101例(94．4％)可检出β-actin DNA表达。36例套细胞淋巴瘤中22例检出t(11;14)易位,对照组无1例检出。去除B-actin和t(11;14)易位均阴性2例,套细胞淋巴瘤中t(11;14)易位检出率为64．7％。(3)36例套细胞淋巴瘤中cyclin D1染色和(或)t(11;14)易位检测阳性病例为29例,总阳性率为80．5％。结论套细胞淋巴瘤石蜡包埋组织中cyclin D1和t(11;14)易位的检测具较高的特异性和可行性,两者的综合应用有助于正确的诊断和鉴别诊断。     

Mantle cell lymphoma: recent insights into pathogenesis, clinical variability, and new diagnostic markers

Mantle cell lymphoma (MCL; previously called centrocytic lymphoma or lymphocytic lymphoma of intermediate differentiation) is a distinct subtype of B-cell lymphoma, accounting for approximately 3%-10% of all lymphoma diagnoses. The name refers to the growth pattern in early disease presentation resembling the normal mantle zone that surrounds the germinal center of the B-cell follicle. The hallmark of MCL is the t(11;14)(q13;q32), resulting in aberrant expression of the CCND1 gene and expression of cyclin D1 in the tumor cells. Expression and genomic profiling of MCL have provided new insight into the pathogenesis and will be summarized in this review. Pitfalls in the differential diagnosis versus B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, cyclin D1-positive diffuse large B-cell lymphoma, hairy cell leukemia, and plasma cell tumors will be discussed, including the usefulness new diagnostic markers SOX11 and CD200. In situ MCL, MCL with an indolent clinical course, and cyclin D1-negative MCL are other topics of this review.